蒺藜苜蓿E3泛素连接酶U-box基因克隆及表达分

引用本文

邵麟惠, 郑兴卫, 李聪. 蒺藜苜蓿E3泛素连接酶U-box基因克隆及表达分. 草业学报, 2016,25(7): 62-72
SHAO Lin-Hui, ZHENG Xing-Wei, LI Cong. Cloning and expression analysis of a U-box gene of E3 ubiquitin ligase from Medicago truncatula . Acta Prataculturae Sinica,2016,25(7): 62-72 复制到剪切板

Permissions

本文属于开放获取期刊。

蒺藜苜蓿E3泛素连接酶U-box基因克隆及表达分

邵麟惠, 郑兴卫, 李聪^*

中国农业科学院北京畜牧兽医研究所,北京100193

*通信作者Corresponding author. E-mail:licong0520@sina.com

作者简介:邵麟惠(1981-),女,甘肃合水人,在读博士。E-mail:shaolinhui@126.com

收稿日期: 2016-03-02

摘要

泛素化在植物生长和发育过程中起重要作用,E3泛素连接酶负责对靶蛋白特异性识别,其中U-box结构的E3泛素连接酶具有调控植物生长发育和免疫反应等功能,并在抗逆性方面发挥作用。目前,关于蒺藜苜蓿U-box家族基因的研究尚未见报道。本研究从蒺藜苜蓿中克隆得到U-box ( MtPUB4)基因,该基因cDNA全长2448 bp,编码815个氨基酸,包括1个U-box结构(C-X2-H-X7-C-X7-C-X2-C-H-X2-H)和5个ARM结构域,属于U-box/ARM类型E3泛素连接酶。构建原核表达载体pET- MtPUB4,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)表明在大肠杆菌中表达了MtPUB4蛋白。荧光定量PCR分析表明 MtPUB4基因在蒺藜苜蓿花中的表达量最高,在根中表达量最低。 MtPUB4基因受到NaCl、聚乙二醇、脱落酸诱导,随着诱导时间增加,表达量呈现出增长趋势。这些结果表明, MtPUB4基因在蒺藜苜蓿生长发育和抗逆性中可能起到重要调节作用,但在低温胁迫中表达量稍微下降,变化不明显。本研究成功构建了pBI121- MtPUB4植物表达载体,为进一步转化拟南芥突变体 Atpub4奠定了基础。

关键词: 蒺藜苜蓿; E3泛素连接酶; U-box; 基因克隆; 表达分析

Cloning and expression analysis of a U-box gene of E3 ubiquitin ligase from Medicago truncatula

SHAO Lin-Hui, ZHENG Xing-Wei, LI Cong^*

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Abstract

The E3 ubiquitin ligase is essential for the specific recognition of target proteins of ubiquitin, and therefore, it plays an important role in plant growth and development. The U-box family of E3 ubiquitin ligases function in regulating plant immune responses and stress resistance. To the best of our knowledge, there have been no reports on U-box family genes in Medicago truncatula. In this study, a U-box gene ( MtPUB4), consisting of a 2448-bp cDNA encoding a putative protein of 815 amino acids, was cloned from M. truncatula. A sequence alignment analysis revealed that the MtPUB4 protein contains a U-box domain (C-X2-H-X7-C-X7-C-X2-C-H-X2-H) and two ARM (Armadillo) regions. Therefore, it was classified as a U-box/ARM E3 ubiquitin-ligase. We constructed a prokaryotic expression vector (pET- MtPUB4), and an SDS-PAGE analysis confirmed that the MtPUB4 protein was successfully expressed in prokaryotic cells. Real-time polymerase chain reaction analyses showed that the transcript levels of MtPUB4 were higher in flowers and lower in roots. Abiotic stresses such as NaCl, polyethylene glycol, and abscisic acid resulted in increased transcript levels of MtPUB4, with higher transcript levels after longer induction times. The transcript levels of MtPUB4 decreased slightly under low-temperature stress. These results indicated that MtPUB4 may be involved in regulating growth, development, and stress resistance in M. truncatula. The construction of the pBI121- MtPUB4 vector has laid the foundation for transformation of Arabidopsis in future studies.

Keyword: Medicago truncatula; E3 ubiquitin-ligase enzyme; U-box; gene cloning; expressing analysis

Show Figures

泛素化是真核生物细胞中重要的蛋白质翻译后修饰的过程, 对生物体生长发育和生物体对周围环境适应性起调控作用, 其中泛素分子是由76个高度保守的氨基酸组成的多肽^[1]。该过程首先是靶蛋白被泛素分子标记, 然后被蛋白酶体识别后降解^[2]。标记靶蛋白的过程由E1泛素活化酶、E2泛素结合酶和E3泛素连接酶共同介导催化完成^[1]。E1泛素活化酶依靠ATP提供能量催化泛素分子与底物蛋白结合, 同时激活泛素分子转移至E2泛素结合酶, 最后通过E3泛素连接酶与赖氨酸残基结合标记靶蛋白^{[3, 4, 5]}。E3泛素连接酶在靶蛋白特异性识别中起关键作用, 依据共价键结合方式, 可分为HECT (homologous to the E6-AP carboxyl terminus)结构域和RING/U-box结构域^[6]。近年来研究表明, E3泛素连接酶参与了各种抗病信号反应途径的调控, 其中U-box蛋白也在抗非生物胁迫和抗病过程中发挥作用^{[7, 8]}。

U-box/ARM蛋白是植物中特有的一类蛋白, 由70多个氨基酸残基构成, 功能域最初在酵母UFD2蛋白中发现, 在多种真核生物中高度保守^[9]。植物U-box蛋白的数量多于其他生物, 目前在拟南芥(Arabidopsis thaliana)中已经鉴定出64个U-box蛋白^[10], 水稻(Oryza sativa)中鉴定出77个U-box蛋白^[11], 蒺藜苜蓿(Medicago truncatula)中鉴定出41个U-box蛋白^[12]。研究表明, 拟南芥pub4基因影响花粉绒毡层细胞生长发育, 在花粉发育的第8~9期开始出现差异变化, 其突变体在22 ℃时花粉绒毡层细胞过度生长膨胀或延迟退化、减小了花粉囊腔空间, 使花粉粒被挤压粘连、无法释放传粉而造成雄性不育, 却在16 ℃的低温时可恢复部分育性, 说明该基因表达方式对温度敏感^[3]。因此, 推测利用此类基因特性可创制出温敏型雄性不育材料。此外, 近年研究人员发现, U-box类基因也参与植物非生物胁迫应答反应, 拟南芥AtPUB22和AtPUB23响应干旱胁迫^[7], AtPUB18和AtPUB19基因受NaCl诱导表达^[13], 辣椒(Capsicum annuum)基因CaPUB1在拟南芥中超表达可以增强植物的耐盐性^[14]。

蒺藜苜蓿是豆科苜蓿属草本^[15]。二倍体(2n=2x=16), 具有生育期短(80~100 d)、基因组小(454~526 Mb)、自花授粉、易于转化、再生时间较短等特点, 被作为豆科模式植物进行研究, 目前已完成全基因组测序^[12]。紫花苜蓿(Medicago sativa)与蒺藜苜蓿同属于苜蓿属植物, 具有相似的遗传特点, 从蒺藜苜蓿中获得的信息对研究其他豆科植物特别是紫花苜蓿具有重要参考价值。目前, 有关蒺藜苜蓿U-box基因家族的研究尚未见报道^[12]。因此, 本试验以蒺藜苜蓿为研究材料, 利用植物U-box基因序列高度保守的特点, 采用RT-PCR方法对蒺藜苜蓿U-box基因编码区序列进行克隆研究, 以期为蒺藜苜蓿U-box基因在花粉发育过程中所起的调控作用和该类基因抗逆特性进行初步探索, 为今后通过基因工程手段选育新种质奠定理论基础。

1 材料与方法

1.1 供试植物与试剂

选取蒺藜苜蓿A17基因型, 蒺藜苜蓿种子由本实验室保存。

RNA提取使用TransZol Up Plus RNA Kit(ER501)试剂盒, cDNA反转录使用TransScript II One-Step gDNA Removal and cDNA Synthesis Supermix(AH311)试剂盒, PCR扩增使用TransStartFastPfu Fly DNA Polymerase酶, 实时荧光定量使用TransStart Top Green qPCR SuperMix试剂盒, 购于北京全式金生物有限公司。DNA maker DL5000、限制性内切酶BamHI、T4 DNA Ligase连接酶, 购于Takara公司; 克隆载体使用pEASY-Uni Seamless Cloning and Assembly Kit(CU101)试剂盒、质粒抽提使用EasyPure Plasmid MiniPrep Kit试剂盒, 原核表达载体使用pEASY-Blunt Simple cloning Kit试剂盒, 胶回收使用 Gel Extraction Kit试剂盒, 分别购于北京全式金生物有限公司和北京康为世纪生物有限公司。大肠杆菌(Escherichia coli)DH5α 购于天根生化科技(北京)有限公司; 根癌农杆菌 (Agrobacterium tumefaciens) AGL1由本实验室保存; pMD18^R-T vector载体购于Takara公司。其他试剂为国产分析纯级。

1.2 实验方法

1.2.1 材料处理

将蒺藜苜蓿种子用酒精消毒后播种于铺有滤纸的培养皿中, 在光照培养箱(温度24 ℃, 光照12 h/d)培养至种子发芽, 将发芽种子用海绵条包裹种植移至盛有MS营养液的水培盘继续培养。20 d左右对幼苗开始处理, 胁迫处理时将水培溶液换成0.1 mol/L NaCl溶液、20%聚乙二醇(PEG)溶液和 1× 10^-4 mol/L ABA溶液, 低温处理的水培溶液换成蒸馏水置于4 ℃冰箱, 分别处理0, 2, 6, 12, 24 h, 各时间点选取叶片组织置于2 mL离心管, 液氮速冻后贮于-80 ℃, 提取总RNA, 用于 Real-time PCR分析。组织特异性分析选取正常栽培条件下结荚期蒺藜苜蓿根、茎、叶、花、荚果。基因克隆选取开花期16 ℃放置48 h的蒺藜苜蓿叶片。

1.2.2 RNA提取及反转录

取蒺藜苜蓿叶片组织100 mg, 按照北京全式金TransZol Plus RNA Kit(ER501)试剂盒说明步骤提取总RNA^[16]。利用紫外分光光度计测定RNA含量, 经琼脂糖凝胶电泳检验, 将质量合格的RNA在-80 ℃条件保存备用。cDNA第一链合成方法, 反应体系为:RNA 1 μ L, 5× TransScript All-in-One SuperMix for qPCR 4 μ L, gDNA Removal 1 μ L, RNase-free Water 14 μ L, 反转录反应体系总体积为20 μ L, 反应条件为:25 ℃ 10 min, 50 ℃ 30 min, 85 ℃ 5 min, 冰浴5 min, -20 ℃条件保存备用。

1.2.3 MtPUB4基因全长克隆及生物信息学分析

从Genbank中下载MtPUB4基因(登录号:XP_013460388.1)序列, 用Primer Premier 5.0和oligo 5.0软件设计并合成扩增引物F1/R1(表1), 以反转录cDNA第一链为模板, 进行PCR扩增。反应体系50 μ L, Template 1 μ L, Primer 各1 μ L, 5× TransStart Buffer 10 μ L, dNTPs 5 μ L; DNA Polymerase 1 μ L, ddH₂O 31 μ L; 反应条件:预变性 95 ℃ 5 min; 95 ℃ 20 s, 55 ℃ 20 s, 72 ℃ 90 s, 35个循环; 延伸72 ℃ 10 min。将PCR扩增产物经凝胶电泳检测合格后胶回收。将胶回收纯化PCR产物, 用pMD^R18-T载体4 ℃条件下连接过夜; 将连接产物转化至DH5α 感受态细胞, 冰浴30 min后, 42 ℃热激30 s, 加入LB培养基于37 ℃恒温摇床振荡培养过夜后, 置于含Amp⁺ 抗性的LB平板中进行筛选, 对所获得的单菌落培养后提取质粒、PCR扩增、用BamHI单酶切鉴定, 重复24次。对菌液进行PCR检测, 选择阳性菌液送北京英俊公司进行DNA测序。测定序列用DNAstar软件进行拼接分析, 得到正确的U-box基因核苷酸序列, 并进行氨基酸序列预测。

利用NCBI网站(http://ncbi.nlm.nih.gov/)ORF Finder和Conservd Domains程序分析核酸和氨基酸序列; 利用在Expasy网站查询(http://www.expasy.org/tools/)预测蛋白功能; 利用Genebank筛选下载其他植物的12种U-box蛋白同源序列, 在MEGA 5.0软件中选择邻近结合法(neighbor-joining)构建进化树, 分析MtPUB4编码的氨基酸序列物种进化关系^[16]。

1.2.4 实时荧光定量PCR检测MtPUB4基因表达量

提取总RNA, 进行反转录合成cDNA第一链, 方法同1.2.2。使用Bio-Rad CFX96 实时定量PCR仪, 采用两步法进行Real-time PCR扩增。基因特异性引物F3/R3(表1), 内参基因选择蒺藜苜蓿ACTIN1, 引物F2/R2(表1)。反应条件:第一步 94 ℃预变性30 s; 第二步 94 ℃ 30 s, 58 ℃ 15 s, 72 ℃ 10 s, 共40个循环。每个样品3次重复。根据实验所得Ct值, 利用Livak等^[17]报道的fold change=2^{-Δ Δ CT}法, 计算蒺藜苜蓿MtPUB4基因在根、茎、叶、花、荚果中的表达量。把根的表达量设为对照, 茎、叶、花、荚果设为4个处理; 不同胁迫处理中, 把0 h表达量设为对照, 2, 6, 12, 24 h设为4个处理, 利用公式进行计算:

Δ Δ C_T=(C_T-gene-C_T-actin)胁迫处理-(C_T-gene-C_T-actin)对照式中, C_T表示循环阈值, C_T-gene表示目标基因达到设定阈值的循环数, C_T-actin表示Actin基因达到设定阈值的循环数, (C_T-gene-C_T-actin)胁迫处理表示处理样品中目标基因经Actin基因校正的循环次数; (C_T-gene-C_T-actin)对照表示对照样品中目标基因经Actin基因校正的循环次数; Δ Δ C_T表示处理样品中目标基因和对照样品中目标基因循环次数的差值。然后再计算出2^{-Δ Δ CT}的相对表达量, 根据计算所得结果绘制柱形图。

1.2.5 蛋白原核表达与鉴定

以蒺藜苜蓿cDNA为模板, 利用引物F4/R4(表1), 进行PCR扩增。取PCR胶回收产物和表达载体pEASY-Blunt Simple进行连接, 将连接产物全部转化Trans1-T1感受态细胞中, 接种SOC(super optimal broth)培养基培养, 涂于LB培养基(含50 μ g/mL Amp⁺)37 ℃过夜培养。次日挑取单菌落, 以引物F5/R5(表1)进行菌液PCR验证, 将合格菌液加入1 mL含Amp⁺抗生素的LB培养基37 ℃培养过夜, 提取合格质粒送公司测序。以构建好的亚克隆载体为模板, 进行PCR扩增目的片段, 引物使用F6/R6(表1), 胶回收PCR产物, 连接pEASY-Blunt E1载体, 转化Trans1-T1感受态细胞, 通过筛选阳性菌液、提取质粒、送交测序, 得到插入目的基因重组子pEASY-MtPUB4。

将阳性克隆菌液20 μ L接种至2 mL LB(含50 μ g/mL的Kan⁺和Amp⁺)培养基中37 ℃过夜培养。取30 μ L过夜培养菌液, 加入至3 mL LB培养基中, 37 ℃震荡培养至OD₆₀₀约0.6; 取部分液体作为未诱导对照组, 余下的分别加入0.1, 0.2, 0.5, 1.0 mmol/L的 IPTG诱导剂作为实验组, 在37 ℃震荡培养。在诱导5 h时取菌体1 mL, 离心10000 r/min× 30 s 收获沉淀, 用100 μ L 1% 磷酸盐缓冲液PBS (phosphate buffer saline) 重悬, 混匀, 100 ℃ 10 min。10000 r/min离心10 min, 取上清进行SDS-PAGE分析。

1.2.6 植物表达载体构建

以克隆测序后的质粒为模板, 使用TransStartKD Plus DNA Polymerase聚合酶, 通过PCR扩增目的片段, 以酶切鉴定引物F7/R7(表1), 用内切限制酶BamHI, 对克隆载体pMD18-T::Mtpub4进行单酶切。酶切体系共200 μ L, 质粒120 μ L, 限制性内切酶4 μ L, 10× CutSmart Buffer 20 μ L, ddH₂O补足至200 μ L。提取单酶切表达载体质粒, 取纯化目的基因PCR产物和pBI121载体, 用pEASY-Uni Seamless Cloning and Assembly Kit (CU101)连接酶连接, 将连接体系置于25 ℃连接2 h; 连接体系共10 μ L, 其中载体3.75 μ L, 目的片段1.25 μ L, 2× Assembly Mix 5 μ L。对于酶切后的表达载体质粒, 取纯化目的产物, 用T4 DNA Ligase连接酶与pBI121载体连接, 将连接体系置于25 ℃连接2 h; 连接体系10 μ L, 其中载体3.0 μ L, 目的片段4 μ L, T4 DNA Ligase 1 μ L, 5× T4 DNA Ligase Buffer 2 μ L。将质粒连接产物转化至DH5α 感受态细胞, 冰浴30 min后, 42 ℃ 热激30 s, 加入400 μ L LB于37 ℃恒温摇床振荡培养, 涂于含Kan⁺ 抗性的LB平板上, 37 ℃过夜培养; 次日挑取单克隆, 进行菌落PCR鉴定; 取鉴定合格菌液, 加2 mL至含Kan⁺抗性的LB培养基中, 37 ℃, 200 r/min转速摇菌过夜, 使用EasyPure Plasmid Mini Prep Kit试剂盒提取质粒; 将质粒进行酶切鉴定, 反应条件为25 ℃ 1 h, 37 ℃ 1 h。取鉴定合格菌液, 送北京英骏公司测序。

表1 引物信息 Table 1 Primers information

2 结果与分析

2.1 目的基因克隆

取蒺藜苜蓿叶片提取总RNA, 凝胶电泳检测显示有28S、18S两个条带, 说明RNA较完整。用紫外分光光度计检测, A₂₆₀/A₂₈₀值为1.877; A₂₆₀/A₂₃₀值为2.054, 说明RNA质量较好(图1A)。将提取RNA反转录合成cDNA第一链; 以cDNA第一链为模板, 以基因特异性引物进行PCR扩增目的片段(图1B), 利用DNAMAN拼接后的全长cDNA为2448 bp。将该基因命名为MtPUB4, NCBI登录号为XP_013460388.1。

	Figure Option View Download New Window
	图1 蒺藜苜蓿RNA提取和MtPUB4基因PCR扩增 A: RNA提取 RNA extraction; B: MtPUB4基因的RT-PCR扩增产物(1~3) Amplification products of MtPUB4 gene by RT-PCR(1-3); M: DNA marker.Fig.1 RNA extraction and U-box E3 ubiquitin-ligating enzyme gene amplication in M. truncatula

2.2 生物信息学分析

2.2.1 保守结构域

运用Expasy网站SMART程序对MtPUB4基因编码的蛋白结构域分析, 结果显示MtPUB4蛋白215~278位氨基酸具有U-box/AEM家族所具备的典型U-box结构, 515~764位氨基酸具有典型的ARM重复结构, 该基因属于U-box家族的一员, 其编码的U-box保守氨基酸序列为FCCPLSLELMTDPVIVASGQTYERAFIKNWIDLGLT VCPKTHQTLAHTNLIPNYTVKALIANW。运用NCBI保守域在线分析, 该蛋白氨基酸序列C端存在U-box结构域, 修饰RING锌指结构, 没有Zn²⁺结合配体, 蛋白N端存在ARM重复结构, 最末端是谷氨酰胺, 可能参与E3泛素化。与其他物种U-box蛋白序列多重比对结果表明, U-box/ARM结构区域保守性较强, 有127个氨基酸完全一致(图2)。

Figure Option
View Download New Window

图2 MtPUB4与其他植物U-box蛋白氨基酸保守区序列多重对比分析
Cicer arietinum:鹰嘴豆; Glycine soja:野大豆; Glycine max:大豆; Phaseolus vulgaris:菜豆; Theobroma cacao:可可; Gossypium arboreum:木本棉; Cucumis sativus:黄瓜; Nicotiana tabacum:烟草; Brassica oleracea:甘蓝; Arabidopsis thaliana:拟南芥; Oryza sativa:水稻。Consensus 表示序列一致性, 黑色表示100%相同, 粉色表示75%相同, 蓝色表示50%相同。Consensus indicates same sequences and are listed below; Black indicates 100% identity; Pink indicates 75% identity; Blue indicates 50% identity.Fig.2 Multiple alignment analysis of MtPUB4 conserved domain with its homologous in other plants

Figure Option
View Download New Window

2.2.2 系统进化

将克隆序列提交NCBI网站BLAST程序进行比对, 选取同源性较高不同物种序列下载, 利用MEGA 5.0软件邻近法程序构建系统进化树^[12]。根据图3显示结果, 克隆序列蒺藜苜蓿MtPUB4与鹰嘴豆(C. arietinum)(XP_0012571337.1)同源性最高, 为89%; 与野生大豆(G. soja)(KHN48396.1)和大豆(G. max)(XP_003551173.1)、菜豆(P. vulgaris)(XP_007146857.1)同源性次之, 都是81%; 与其他科属植物可可(T. cacao)(XP_007047436.1)、木本棉(G. raimondii)(KJB48641.1)、黄瓜(C. sativus)(XP_004149702.1)、烟草(N. tabacum)(AAO61490.1)、甘蓝(B. oleracea)(XP_013605017.1)、拟南芥(A. thaliana)(NP_179895.6)和水稻(O. sativa)(AAT94161.1)等植物同源性较低, 分别是71%, 69%, 65%, 58%, 56%, 56%和54%。

	Figure Option View Download New Window
	图3 蒺藜苜蓿U-box E3泛素蛋白MtPUB4进化树分析Fig.3 Phylogenetic tree analysis of U-box E3 ubiquitin-ligating enzyme MtPUB4 proteins in M. truncatula

2.3 蒺藜苜蓿MtPUB4基因组织特异性表达

蒺藜苜蓿MtPUB4基因在不同组织中的相对表达量见图4。结果表明, MtPUB4在花中表达量最多, 其次是叶, 根中表达量最少。以蒺藜苜蓿根中MtPUB4基因表达量为对照, 花中表达量是根中表达量的26.68倍, 叶中表达量是根中15.45倍, 荚果中表达量是根中的5.60倍, 茎中表达量是根中的4.82倍。

	Figure Option View Download New Window
	图4 MtPUB4基因在蒺藜苜蓿不同组织中的相对表达量Fig.4 The relative expression of MtPUB4 gene in different tissues of M. truncatula

2.4 蒺藜苜蓿MtPUB4基因在非生物胁迫下的表达量

蒺藜苜蓿MtPUB4基因分别受到NaCl、PEG、ABA和低温诱导0, 2, 6, 12, 24 h的相对表达量见图5。结果显示, MtPUB4基因在NaCl胁迫诱导下, 6 h时迅速上升, 是2 h时表达量的3倍, 12 h时达到峰值, 24 h下降(图5A)。在PEG胁迫诱导下2 h以内变化较小, 6 h表达量增到2 h的4倍, 达到峰值, 与NaCl胁迫下变化近似, 但是12 h时开始下降, 24 h时表达量低于0 h时表达量(图5B)。在低温处理后, 2 h时表达量稍微有所下降, 6 h时表达量与2 h表达量接近, 12 h时持续下降, 24 h时恢复至6 h水平(图5C)。在ABA诱导12 h后, 表达量稍有降低, 随后迅速上升, 24 h时表达量接近0 h的5倍(图5D)。除了低温胁迫MtPUB4基因表达量变化稍有降低, 表达量变化不明显外, 其余3种处理方式表达量都出现数倍上升, 这说明MtPUB4基因对盐、干旱和ABA胁迫响应, 可能参与这些逆境的信号调控。

The relative expression of *MtPUB*4 gene treated by NaCl, PEG, ABA and 4 ℃
	Figure Option View Download New Window
	图5 MtPUB4基因在NaCl、PEG、4 ℃低温和ABA处理后的相对表达量A: NaCl 处理 NaCl treatment; B: PEG处理 PEG treatment ; C: 4 ℃ 低温处理 4 ℃ treatment; D:脱落酸处理 ABA(abscisic acid) treatmentFig. 5

2.5 MtPUB4基因蛋白原核表达

将目的片段连接pEASY-Blunt Simple载体, 转化至大肠杆菌BL21(DE3), 经测序验证序列正确, 表明蛋白表达载体构建成功。将含有阳性质粒的大肠杆菌表达菌BL21(DE3)pLysS Chemically Competent Cell株系, 用不同浓度的IPTG诱导表达, 上样浓度为0.1, 0.2, 0.5和1.0 mmol/L。结果显示, 含有MtPUB4片段的表达载体菌体在诱导后出现特异性条带, 各个浓度对蛋白表达影响不明显。该蛋白在90 kDa条带处, 与预期结果88.36 kDa相符(图6)。说明该蛋白在大肠杆菌中成功诱导, 可用于进一步蛋白表达研究。

	Figure Option View Download New Window
	图6 MtPUB4融合蛋白SDS-PAGE电泳考马斯亮蓝染色 1~5 :0, 0.1, 0.2, 0.5和1.0 mmol/L IPTG诱导的菌体蛋白。 1-5 indicate 0, 0.1, 0.2, 0.5 and 1.0 mmol/L IPTG induction bacterial protein; M:蛋白标记 Protein marker.Fig.6 Coomassie-stained SDS-PAGE gel illustrating MtPUB4 His-tag fusion protein expression in E. coli

2.6 植物表达载体构建

将植物表达载体pBI121和克隆载体分别酶切, 回收相应片段纯化连接、转化、挑取单菌落、提取质粒, 构建MtPUB4基因植物表达载体。经过BamHI酶切鉴定, 得到与预期大小一致的目的片段(图7)。鉴定为阳性的质粒送公司测序后拼接, 与基因序列一致, 说明成功构建植物表达pBI121-MtPUB4。

	Figure Option View Download New Window
	图7 植物表达载体PCR和酶切验证 1~2:表达载体pBI121质粒提取结果。1-2: Results of vector pBI121 plasmid extraction; 3~4:质粒单酶切后结果。3-4: Results of single enzyme digestion; M:DL5000 DNA marker.Fig.7 Identification of expression vector by enzyme digestion

3 讨论

U-box E3泛素连接酶基因在植物生长发育和抗逆过程中起着重要作用。目前E3 泛素连接酶U-box的研究主要集中在拟南芥、水稻、烟草等模式植物中。本研究得到的蒺藜苜蓿MtPUB4基因的氨基酸序列中(图3), 在C端存在Ring super family U-box结构域, 有Zn指结构40个残基结合2个Zn原子, 由半胱氨酸和组氨酸组成, 可能参与蛋白质与蛋白质之间的相互反应, Hatakeyama等^[8]认为这类蛋白与序列复制、信号传导和发育有关; 在N端有连续5个ARM重复结构, 末端是谷氨酰胺, 推测参与E3泛素化。有学者认为ARM结构域蛋白质在植物生长发育和激素应答等方面起重要作用^{[9, 18]}。在某些植物U-box蛋白中, 如拟南芥中ARC1(Arm Repeat Containing 1), 其C端含有一个6~7个ARM重复结构功能域, 中间含有一个U-box功能域, 是SRK激酶下游信号的传递因子, 能在柱头乳突细胞中特异性表达, 能促进花粉水合、萌发和花粉管生长的雌性亲和因子^[19]。在水稻中发现的SPL11^{[20, 21, 22]}、烟草中的ACER276^[23]、拟南芥中的AtPUB17等U-box基因都与植物抗病性有关^[24]; 马铃薯(Solanum tuberosum)中发现的U-box基因PHOR1(photoperiod-responsive1)参与GA信号转导, 是GA信号转导途径中的正调控因子^[25]; 烟草中的NtPUB4参与了植物发育和细胞分裂素信号转导^[26]; 拟南芥AtPUB4的U-box/ARM功能域缺失能使绒毡层结构肥大最终导致植物雄性不育^[3]; 蒺藜苜蓿中未见报道。通过构建系统进化树结果表明, 它们与同科属植物同源性很高, 和其他科属的植物同源性较低, 这与其他学者在其他物种间同源性分析结论基本一致^{[16, 27]}, 说明同源关系较近的植物中基因保守性更强。依据属内同源性较高的理论, 说明U-box/ARM蛋白家族基因进化上也比较保守, 其氨基酸序列相似度可在一定程度上说明了不同物种亲缘关系远近, 本研究中的蒺藜苜蓿MtPUB4基因与拟南芥AtPUB4基因、烟草NtPUB4的同源性较低, 但是在同一类型进化枝上, 这可能与拟南芥与烟草基因组较小有关系。

前人研究表明U-box E3连接酶可调控植物多个生理代谢途径, Samuel等^[28]研究发现, 拟南芥中的U-box/ARM蛋白家族与拟南芥SD1受体蛋白激酶亚家族相互结合, 通过酵母双杂交实验表明, 两个受体结合可使U-box/ARM蛋白的ARM功能域磷酸化, 从而参与调控植物生理过程。Kim等^[26]研究认为, 烟草的NtPUB4参与CHRK1介导的植物发育及细胞分裂素信号转导过程。据报道, CHRK1是烟草内的一种新型类受体激酶, 参与调控发育信号转导及内源细胞分裂素平衡, 且NtPUB4在烟草的花器官大量表达, 在苗、根、茎、叶中少量表达。本研究中, 蒺藜苜蓿Mtpub4基因也在花中大量表达, 与NtPUB4、ARC1有相似的结构域, 因此推测它们具有相似的功能。而CHRK1与SRK类受体激酶聚类在一起, 推测NtPUB4与ARC1功能相似^[26]。Samuel等^[28]在拟南芥中研究发现, 一些U-box/ARM基因受到NaCl胁迫诱导表达, 基因表达量会上调。以上结果表明U-box基因在抗性方面也可能发挥作用。本实验增加了干旱、低温和ABA诱导方面的研究, 通过模拟各种胁迫条件, MtPUB4基因在受到NaCl、PEG和ABA胁迫诱导后, 基因表达量都数倍提高, 此基因在NaCl、PEG和ABA胁迫反应中也可能参与调控反应, 但在低温胁迫中, 表达量稍微下降, 变化不明显。

雄性不育与一些基因突变有关, 产生这些基因突变的主要原因包括光周期、光强度、湿度和温度等环境因素的改变。目前, 利用这种现象人工筛选突变体, 通过两系配套法培育不育系已在杂交作物的生产上成功应用^[29]。在拟南芥中, 突变体Atpub4在16 ℃中可恢复部分育性, 主要是花粉囊中的花粉粒可成功授粉。目前尚无蒺藜苜蓿U-box E3泛素连接酶蛋白的相关研究^[30], 我们通过全基因组U-box结构域分析得出, 蒺藜苜蓿MtPUB32可能与ARK2共同调控植株侧根发育, MtPUB26可能与干旱胁迫相关^[12]。通过对克隆蒺藜苜蓿MtPUB4序列分析, 得出它与拟南芥AtPUB4蛋白间有共同保守域结构, 利用生物信息学相关软件分析基因序列特征、功能蛋白结构、基因进化关系, 发现克隆基因属于植物U-box家族成员, 具备1个U-box结构域和5个ARM重复结构。本研究为进一步了解U-box基因如何调控蒺藜苜蓿的生长发育, 以及如何参与逆境胁迫信号转导, 揭示植物生长发育过程中的一些机制提供理论基础。

4 结论

本实验通过RT-PCR技术, 克隆了蒺藜苜蓿U-box E3泛素连接酶基因, 对其核苷酸、氨基酸序列对比分析, 检测相对表达量变化规律, 进行原核表达并提取了MtPUB4蛋白, 同时成功构建植物表达载体。为后期筛选蒺藜苜蓿雄性不育基因、将其转化至拟南芥Atpub4雄性不育突变体材料进行遗传互补提供了候选基因。

The authors have declared that no competing interests exist.

参考文献

View Option

[1]	Zeng L . , Park C . , Venu R . , et al. Classification, expression pattern, and E3 ligase activity assay of rice U-box-containing proteins. Molecular Plant, 2008, 1(5): 800-815. [本文引用:2]
[2]	Chen J . , Zheng L . , Wang B . , et al. The nonproteolytic functions mediated by ubiquitylation. Progress in Biochemistry and Biophysics, 2012, 7: 613-621. [本文引用:1]
[3]	Wang . , Lu . , Jiang . , et al. The Arabidopsis U-box/ARM repeat E3 ligase AtPUB4 influences growth and degeneration of tapetal cells, and its mutation leads to conditional male sterility. The Plant Journal: for Cell and Molecular Biology, 2013, 74(3): 511-523. [本文引用:3]
[4]	Gonzalez-Lamothe . , Tsitsigiannis D . , Ludwig A . , et al. The U-box protein CMPG1 is required for efficient activation of defense mechanisms triggered by multiple resistance genes in tobacco and tomato. Plant Cell, 2006, 18(4): 1067-1083. [本文引用:1]
[5]	Yang J . , Zhang . , Wang Z . , et al. Recent progresses in the regulation mechanism of E3 ligases in plant disease resistance. Plant Protection, 2015, 4: 1-8, 28. [本文引用:1]
[6]	Mudgil . , Shiu S . , Stone S . , et al. A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family. Plant Physiology, 2004, 134(1): 59-66. [本文引用:1]
[7]	Cho S . , Ryu M . , Song . , et al. Arabidopsis PUB22 and PUB23 are homologous U-Box E3 ubiquitin ligases that play combinatory roles in response to drought stress. Plant Cell, 2008, 20(7): 1899-1914. [本文引用:2]
[8]	Hatakeyama . , Yada . , Matsumoto . , et al. U-box proteins as a new family of ubiquitin-protein ligases. The Journal of Biological Chemistry, 2001, 276(35): 33111-33120. [本文引用:2]
[9]	Hur Y . , Yi Y . , Lee J . , et al. Molecular cloning and characterization of OsUPS, a U-box containing E3 ligase gene that respond to phosphate starvation in rice (Oryza sativa). Molecular Biology Reports, 2012, 39(5): 5883-5888. [本文引用:2]
[10]	Lu Y . , Guan . , Li C. Advance in the study on U-box proteins of Arabidopsis thaliana. Molecular Plant Breeding, 2008, 6: 941-948. [本文引用:1]
[11]	Chen . , Li L . , Bai . , et al. Expression analysis of Rice U-Box proteins at different developmental stages. Progress in Biochemistry and Biophysics, 2009, 36(9): 1208-1214. [本文引用:1]
[12]	Zheng X . , Shao L . , Li C. Genome-wide screening and characterization of the U-box gene family in Medicago truncatula. Acta Prataculturae Sinica, 2015, 24(8): 130-141. [本文引用:5]
[13]	Bergler . , Hoth S. Plant U-box armadillo repeat proteins AtPUB18 and AtPUB19 are involved in salt inhibition of germination in Arabidopsis. Plant Biology, 2011, 13(5): 725-730. [本文引用:1]
[14]	Cho S . , Chung H . , Ryu M . , et al. Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog. Plant Physiology, 2006, 142(4): 1664-1682. [本文引用:1]
[15]	Wei Z . , Gai J Y. Model legume: Medicago truncatula. Acta Prataculturae Sinica, 2008, 17(1): 114-120. [本文引用:1]
[16]	Zou . , Zhang . , Wu M . , et al. Cloning and analysis of the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPC) gene from Solanum tuberosum and Arabidopsis thaliana. Acta Prataculturae Sinica, 2014, 23(1): 239-247. [本文引用:3]
[17]	Livak K . , Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 2001, 25(4): 402-408. [本文引用:1]
[18]	Mazzucotelli . , Belloni . , Marone . , et al. The E3 ubiquitin ligase gene family in plants: regulation by degradation. Current Genomics, 2006, 7(8): 509-522. [本文引用:1]
[19]	Nasrallah J . , Nasrallah M E. Robust self-incompatibility in the absence of a functional ARC1 gene in Arabidopsis thaliana. The Plant Cell, 2014, 26(10): 3838-3841. [本文引用:1]
[20]	Liu . , Park C . , He . , et al. The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice. PLoS Pathogens, 2015, 11(2): e1004629. [本文引用:1]
[21]	Vega-Sanchez M . , Zeng . , Chen . , et al. SPIN1, a K homology domain protein negatively regulated and ubiquitinated by the E3 ubiquitin ligase SPL11, is involved in flowering time control in rice. The Plant Cell, 2008, 20(6): 1456-1469. [本文引用:1]
[22]	Liu . , Li . , Ning . , et al. The U-box E3 ligase SPL11/PUB13 is a convergence point of defense and flowering signaling in plants. Plant Physiology, 2012, 160(1): 28-37. [本文引用:1]
[23]	Yang C . , GonzAilez-Lamothe, Rocio, et al. The E3 ubiquitin ligase activity of Arabidopsis PLANT U-BOX17 and its functional tobacco homolog ACRE276 are required for cell death and defense. The Plant Cell, 2006, 18(4): 1084-1098. [本文引用:1]
[24]	He . , McLellan H, Boevink P C, et al. U-box E3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans. Journal of Experimental Botany, 2015, 66(11): 3189-3199. [本文引用:1]
[25]	Amador . , Monte . , Garcia-Martinez J L, et al. Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo. Cell, 2001, 106(3): 343-354. [本文引用:1]
[26]	Kim . , Cho H . , Kim D . , et al. CHRK1, a chitinase-related receptor-like kinase, interacts with NtPUB4, an armadillo repeat protein, in tobacco. Biochimica et Biophysica Acta, 2003, 1651(1-2): 50-59. [本文引用:3]
[27]	Niu J . , Zhang J . , Wang W . , et al. Cloning and sequence analysis of terminase gene of glycoalkaloid biosynthesis metabolismic pathway in potato. Acta Prataculturae Sinica, 2012, 21(3): 106-116. [本文引用:1]
[28]	Samuel M . , Mudgil . , Salt J . , et al. Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis. Plant Physiology, 2008, 147(4): 2084-2095. [本文引用:2]
[29]	Zhou . , Liu . , Li . , et al. Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA. Cell Research, 2012, 22(4): 649-660. [本文引用:1]
[30]	Wiborg . , O’Shea C, Skriver K. Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases. Biochemistry Journal, 2008, 413(3): 447-457. [本文引用:1]
[2]	陈季武, 郑丽娜, 王帮正, 等. 泛素化介导的非蛋白质降解功. 生物化学与生物物理进展, 2012, (7): 613-621. [本文引用:1]
[5]	杨玖霞, 张浩, 王志龙, 等. E3泛素连接酶调控植物抗病分子机理研究进. 植物保护, 2015, 4: 1-8, 28. [本文引用:1]
[10]	鲁玉清, 关宁, 李聪. 拟南芥U-box蛋白的研究进. 分子植物育种, 2008, 6: 941-948. [本文引用:1]
[12]	郑兴伟, 邵麟惠, 李聪. 蒺藜苜蓿全基因组中U-box基因家族的筛选与特征分. 草业学报, 2015, 24(8): 130-141. [本文引用:5]
[15]	魏臻武, 盖钧镒. 豆科模式植物-蒺藜苜. 草业学报, 2008, 17(1): 114-120. [本文引用:1]
[16]	邹雪, 张烨, 吴明阳, 等. 马铃薯和拟南芥GAPC酶基因的克隆及分. 草业学报, 2014, 23(1): 239-247. [本文引用:3]
[27]	牛继平, 张金文, 王旺田, 等. 马铃薯SGAs合成代谢途径末端SGT酶基因克隆及序列分. 草业学报, 2012, 21(3): 106-116. [本文引用:1]

2008

0.0

. 2008, 1(5):800-815 DOI:10.1093/mp/ssn044

Classification, expression pattern, and E3 ligase activity assay of rice U-box-containing proteins

Zeng L , Park C , Venu R

Department of Plant Pathology, The Ohio State University, Columbus, OH 43210, USA.

Ubiquitin ligases play a central role in determining the specificity of the ubiquitination system by selecting a myriad of appropriate candidate proteins for modification. The U-box is a recently identified, ubiquitin ligase activity-related protein domain that shows greater presence in plants than in other organisms. In this study, we identified 77 putative U-box proteins from the rice genome using a battery of whole genome analysis algorithms. Most of the U-box protein genes are expressed, as supported by the identification of their corresponding expressed sequence tags (ESTs), full-length cDNAs, or massively parallel signature sequencing (MPSS) tags. Using the same algorithms, we identified 61 U-box proteins from the Arabidopsis genome. The rice and Arabidopsis U-box proteins were classified into nine major classes based on their domain compositions. Comparison between rice and Arabidopsis U-box proteins indicates that the majority of rice and Arabidopsis U-box proteins have the same domain organizations. The inferred phylogeny established the homology between rice and Arabidopsis U-box/ARM proteins. Cell death assay using the rice protoplast system suggests that one rice U-box gene, OsPUB51, might act as a negative regulator of cell death signaling. In addition, the selected U-box proteins were found to be functional E3 ubiquitin ligases. The identification and analysis of rice U-box proteins hereby at the genomic level will help functionally characterize this class of E3 ubiquitin ligase in the future.

PMID: 19825583

... 泛素化是真核生物细胞中重要的蛋白质翻译后修饰的过程,对生物体生长发育和生物体对周围环境适应性起调控作用,其中泛素分子是由76个高度保守的氨基酸组成的多肽^[1] ...

... 标记靶蛋白的过程由E1泛素活化酶、E2泛素结合酶和E3泛素连接酶共同介导催化完成^[1] ...

2012

0.0

... 该过程首先是靶蛋白被泛素分子标记,然后被蛋白酶体识别后降解^[2] ...

2013

0.0

. 2013, 74(3):511-523 DOI:10.1111/tpj.12146

The Arabidopsis U-box/ARM repeat E3 ligase AtPUB4 influences growth and degeneration of tapetal cells, and its mutation leads to conditional male sterility

Wang , Lu , Jiang

Department of Biology, Hong Kong Baptist University, 224 Waterloo Rd, Hong Kong, China.

Pollen formation is a complex developmental process that has been extensively investigated to unravel underlying fundamental developmental mechanisms and for genetic manipulation of the male-sterility trait for hybrid crop production. Here we describe identification of AtPUB4, a U-box/ARM repeat-containing E3 ubiquitin ligase, as a novel player in male fertility in Arabidopsis. Loss of AtPUB4 function causes hypertrophic growth of the tapetum layer. The Atpub4 mutation also leads to incomplete degeneration of the tapetal cells and strikingly abnormal exine structures of pollen grains. As a result, although the Atpub4 mutant produces viable pollen, the pollen grains adhere to each other and to the remnants of incompletely degenerated tapetal cells, and do not properly disperse from dehisced anthers for successful pollination. We found that the male-sterility phenotype caused by the Atpub4 mutation is temperature-dependent: the mutant plants are sterile when grown at 22°C but are partially fertile at 16°C. Our study also indicates that the AtPUB4-mediated pathway acts in parallel with the brassinosteroid pathway in controlling developmental fates of the tapetal cells to ensure male fertility.

PMID: 23398263

... E1泛素活化酶依靠ATP提供能量催化泛素分子与底物蛋白结合,同时激活泛素分子转移至E2泛素结合酶,最后通过E3泛素连接酶与赖氨酸残基结合标记靶蛋白^[3,4,5] ...

... 研究表明,拟南芥pub4基因影响花粉绒毡层细胞生长发育,在花粉发育的第8~9期开始出现差异变化,其突变体在22 ℃时花粉绒毡层细胞过度生长膨胀或延迟退化、减小了花粉囊腔空间,使花粉粒被挤压粘连、无法释放传粉而造成雄性不育,却在16 ℃的低温时可恢复部分育性,说明该基因表达方式对温度敏感^[3] ...

... 拟南芥AtPUB4的U-box/ARM功能域缺失能使绒毡层结构肥大最终导致植物雄性不育^[3] ...

2006

0.0

. 2006, 18(4):1067-1083

The U-box protein CMPG1 is required for efficient activation of defense mechanisms triggered by multiple resistance genes in tobacco and tomato

Gonzalez-Lamothe , Tsitsigiannis D , Ludwig A

Sainsbury Laboratory, John Ines Centre, Norwich NR4 7UH, UK.

We previously identified three Avr9/Cf-9 Rapidly Elicited (ACRE) genes essential for Cf-9- and Cf-4-dependent hypersensitive response (HR) production in Nicotiana benthamiana. Two of them encode putative E3 ubiquitin ligase components. This led us to investigate other ACRE genes associated with the ubiquitination pathway. ACRE74 encodes a U-box E3 ligase homolog, highly related to parsley (Petroselinum crispum) CMPG1 and Arabidopsis thaliana PLANT U-BOX20 (PUB20) and PUB21 proteins, and was called Nt CMPG1. Transcript levels of Nt CMPG1 and the homologous tomato (Solanum lycopersicum) Cmpg1 are induced in Cf9 tobacco (Nicotiana tabacum) and Cf9 tomato after Avr9 elicitation. Tobacco CMPG1 possesses in vitro E3 ligase activity. N. benthamiana plants silenced for Nt CMPG1 show reduced HR after Cf-9/Avr9 elicitation, while overexpression of Nt CMPG1 induces a stronger HR in Cf9 tobacco plants after Avr9 infiltration. In tomato, silencing of Cmpg1 decreased resistance to Cladosporium fulvum. Overexpression of epitope-tagged tobacco CMPG1 mutated in the U-box domain confers a dominant-negative phenotype. We also show that Nt CMPG1 is involved in the Pto/AvrPto and Inf1 responses. In summary, we show that the E3 ligase Nt CMPG1 is essential for plant defense and disease resistance.

PMID: 16531490

2015

0.0

. 2015, 4:1-8, 28

E3泛素连接酶调控植物抗病分子机理研究进

杨玖霞, 张浩, 王志龙

Biotic stress is an important factor that affects crop production in China. It is also one of the most widely studied areas in plant sciences. Degradation mediated by the ubiquitin proteasome system (UPS) is one of the most sophisticated regulation systems in eukaryotes, which is involved in plant growth and development and in response to abiotic and biotic stresses. E3 ligase is considered as a key enzyme in the UPS due to its specific interactions with its substrates. Based on the differences in structure and function, E3 ligases can be divided into four main classes. In this paper, we review the recent progresses in the regulation mechanism of E3 ligases in plant disease resistance and propose new research directions.

生物胁迫是影响我国农作物生产的重要因素,也是当前植物界研究方向涉及最为广泛的领域之一。由泛素介导的降解途径是生物体内最为精细的调控体系,涉及对生物体的生长发育以及生物体对周围环境适应的调控等过程。E3泛素连接酶因对底物有特异性识别作用,被认为是泛素化过程中最重要的组成部分。依据其结构和功能的不同可以将E3泛素连接酶分为4个家族。越来越多的研究表明这些不同的E3家族成员可以参与植物抗病免疫反应的各个过程。本文在简要概括E3泛素连接酶分类的基础上综述了目前E3泛素连接酶参与调控植物抗病害方面研究进展,并对今后研究方向进行了展望,以期对抗病机理解析及抗病品种研发提供新思路。

2004

0.0

. 2004, 134(1):59-66

A large complement of the predicted Arabidopsis ARM repeat proteins are members of the U-box E3 ubiquitin ligase family

Mudgil , Shiu S , Stone S

Department of Botany, University of Toronto, Toronto, Ontario, Canada M5S 3B2.

The Arabidopsis genome was searched to identify predicted proteins containing armadillo (ARM) repeats, a motif known to mediate protein-protein interactions in a number of different animal proteins. Using domain database predictions and models generated in this study, 108 Arabidopsis proteins were identified that contained a minimum of two ARM repeats with the majority of proteins containing four to eight ARM repeats. Clustering analysis showed that the 108 predicted Arabidopsis ARM repeat proteins could be divided into multiple groups with wide differences in their domain compositions and organizations. Interestingly, 41 of the 108 Arabidopsis ARM repeat proteins contained a U-box, a motif present in a family of E3 ligases, and these proteins represented the largest class of Arabidopsis ARM repeat proteins. In 14 of these U-box/ARM repeat proteins, there was also a novel conserved domain identified in the N-terminal region. Based on the phylogenetic tree, representative U-box/ARM repeat proteins were selected for further study. RNA-blot analyses revealed that these U-box/ARM proteins are expressed in a variety of tissues in Arabidopsis. In addition, the selected U-box/ARM proteins were found to be functional E3 ubiquitin ligases. Thus, these U-box/ARM proteins represent a new family of E3 ligases in Arabidopsis.

PMID: 14657406

... E3泛素连接酶在靶蛋白特异性识别中起关键作用,依据共价键结合方式,可分为HECT (homologous to the E6-AP carboxyl terminus)结构域和RING/U-box结构域^[6] ...

2008

0.0

. 2008, 20(7):1899-1914 DOI:10.1105/tpc.108.060699

Arabidopsis PUB22 and PUB23 are homologous U-Box E3 ubiquitin ligases that play combinatory roles in response to drought stress

Cho S , Ryu M , Song

Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea.

Ubiquitination is involved in diverse cellular processes in higher plants. In this report, we describe Arabidopsis thaliana PUB22 and PUB23, two homologous U-box-containing E3 ubiquitin (Ub) ligases. The PUB22 and PUB23 genes were rapidly and coordinately induced by abiotic stresses but not by abscisic acid. PUB22- and PUB23-overexpressing transgenic plants were hypersensitive to drought stress. By contrast, loss-of-function pub22 and pub23 mutant plants were significantly more drought-tolerant, and a pub22 pub23 double mutant displayed even greater drought tolerance. These results indicate that PUB22 and PUB23 function as negative regulators in the water stress response. Yeast two-hybrid, in vitro pull-down, and in vivo coimmunoprecipitation experiments revealed that PUB22 and PUB23 physically interacted with RPN12a, a subunit of the 19S regulatory particle (RP) in the 26S proteasome. Bacterially expressed RPN12a was effectively ubiquitinated in a PUB-dependent fashion. RPN12a was highly ubiquitinated in 35S:PUB22 plants, but not in pub22 pub23 double mutant plants, consistent with RPN12a being a substrate of PUB22 and PUB23 in vivo. In water-stressed wild-type and PUB-overexpressing plants, a significant amount of RPN12a was dissociated from the 19S RP and appeared to be associated with small-molecular-mass protein complexes in cytosolic fractions, where PUB22 and PUB23 are localized. Overall, our results suggest that PUB22 and PUB23 coordinately control a drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis.

PMID: 18664614

... 近年来研究表明,E3泛素连接酶参与了各种抗病信号反应途径的调控,其中U-box蛋白也在抗非生物胁迫和抗病过程中发挥作用^[7,8] ...

... 此外,近年研究人员发现,U-box类基因也参与植物非生物胁迫应答反应,拟南芥AtPUB22和AtPUB23响应干旱胁迫^[7],AtPUB18和AtPUB19基因受NaCl诱导表达^[13],辣椒(Capsicum annuum)基因CaPUB1在拟南芥中超表达可以增强植物的耐盐性^[14] ...

2001

0.0

. 2001, 276(35):33111-33120

U-box proteins as a new family of ubiquitin-protein ligases

Hatakeyama , Yada , Matsumoto

Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

The U box is a domain of approximately 70 amino acids that is present in proteins from yeast to humans. The prototype U box protein, yeast Ufd2, was identified as a ubiquitin chain assembly factor that cooperates with a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin-protein ligase (E3) to catalyze ubiquitin chain formation on artificial substrates. E3 enzymes are thought to determine the substrate specificity of ubiquitination and have been classified into two families, the HECT and RING finger families. Six mammalian U box proteins have now been shown to mediate polyubiquitination in the presence of E1 and E2 and in the absence of E3. These U box proteins exhibited different specificities for E2 enzymes in this reaction. Deletion of the U box or mutation of conserved amino acids within it abolished ubiquitination activity. Some U box proteins catalyzed polyubiquitination by targeting lysine residues of ubiquitin other than lysine 48, which is utilized by HECT and RING finger E3 enzymes for polyubiquitination that serves as a signal for proteolysis by the 26 S proteasome. These data suggest that U box proteins constitute a third family of E3 enzymes and that E4 activity may reflect a specialized type of E3 activity.

PMID: 11435423

... 近年来研究表明,E3泛素连接酶参与了各种抗病信号反应途径的调控,其中U-box蛋白也在抗非生物胁迫和抗病过程中发挥作用^[7,8] ...

... 本研究得到的蒺藜苜蓿MtPUB4基因的氨基酸序列中(图3),在C端存在Ring super family U-box结构域,有Zn指结构40个残基结合2个Zn原子,由半胱氨酸和组氨酸组成,可能参与蛋白质与蛋白质之间的相互反应,Hatakeyama等^[8]认为这类蛋白与序列复制、信号传导和发育有关 ...

2012

0.0

. 2012, 39(5):5883-5888 DOI:10.1007/s11033-011-1399-5

Molecular cloning and characterization of OsUPS, a U-box containing E3 ligase gene that respond to phosphate starvation in rice (Oryza sativa)

Hur Y , Yi Y , Lee J

College of Life Science and Natural Resources, Dong-A University, Busan, 604-714, Korea.

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41-79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P(i)). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P(i) signaling pathway through the ubiquitin-26S proteasome system.

PMID: 22201023

... U-box/ARM蛋白是植物中特有的一类蛋白,由70多个氨基酸残基构成,功能域最初在酵母UFD2蛋白中发现,在多种真核生物中高度保守^[9] ...

... 有学者认为ARM结构域蛋白质在植物生长发育和激素应答等方面起重要作用^[9,18] ...

2008

0.0

... 植物U-box蛋白的数量多于其他生物,目前在拟南芥(Arabidopsis thaliana)中已经鉴定出64个U-box蛋白^[10],水稻(Oryza sativa)中鉴定出77个U-box蛋白^[11],蒺藜苜蓿(Medicago truncatula)中鉴定出41个U-box蛋白^[12] ...

2009

0.0

2015

0.0

. 2015, 24(8):130-141 DOI:10.11686/cyxb2015102

蒺藜苜蓿全基因组中U-box基因家族的筛选与特征分

郑兴伟, 邵麟惠, 李聪

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

The U-box domain genes typically exist in multiple variants within plant genomes, and most of them belong to the ubiquitin/proteasome system as E 3 ubiquitin ligase. They play key roles in the regulation of growth, in development, in reproduction as well as in response to environmental stress. In this study, bioinformatics methods were applied to identify U-box family genes of M. truncatula through whole genome screening. A phylogenetic tree was created using the MEGA6 program. Gene structure and chromosome location were defined by GSDS and Mapinspect. A total of 41 U-box genes distributed unevenly on 8 chromosomes were identified from M. truncatula . The U-box proteins were classified into six major classes based on their motif compositions and phylogeny relationships. The expression profiles of M. truncatula U-box genes showed characteristics of tissue specificity and response to salt, drought and nitrogen stress. These results will be useful for future functional analyses of the U-box family genes in M. truncatula .

植物基因组中广泛存在U-box基因,其编码蛋白大部分作为泛素系统中决定底物特异性识别的E 3 泛素连接酶,广泛地调控植物生长生殖发育以及响应逆境胁迫等过程。本文利用蒺藜苜蓿基因组数据库,通过生物信息学手段,鉴定蒺藜苜蓿U-box家族基因;采用MEGA6软件进行系统进化树分析;通过GSDS在线工具和Mapinspect软件进行基因结构及染色体定位分析;利用已有的蒺藜苜蓿芯片数据进行组织表达和胁迫响应表达分析。结果表明,蒺藜苜蓿基因组中含有41个U-box基因,不均匀地分布于蒺藜苜蓿的8条染色体上。根据结构域组成和系统进化分析将这些U-box蛋白分成6类。基因表达模式分析发现,该家族成员的表达具有一定的组织特异性,并能响应盐、干旱和氮素胁迫。这些研究结果为蒺藜苜蓿U-box基因家族的功能分析奠定了基础。

... 二倍体(2n=2x=16),具有生育期短(80~100 d)、基因组小(454~526 Mb)、自花授粉、易于转化、再生时间较短等特点,被作为豆科模式植物进行研究,目前已完成全基因组测序^[12] ...

... 目前,有关蒺藜苜蓿U-box基因家族的研究尚未见报道^[12] ...

... 0软件邻近法程序构建系统进化树^[12] ...

... 目前尚无蒺藜苜蓿U-box E3泛素连接酶蛋白的相关研究^[30],我们通过全基因组U-box结构域分析得出,蒺藜苜蓿MtPUB32可能与ARK2共同调控植株侧根发育,MtPUB26可能与干旱胁迫相关^[12] ...

2011

0.0

. 2011, 13(5):725-730 DOI:10.1111/j.1438-8677.2010.00431.x

Plant U-box armadillo repeat proteins AtPUB18 and AtPUB19 are involved in salt inhibition of germination in Arabidopsis

Bergler , Hoth S

Molekulare Pflanzenphysiologie, Department Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.

Plant U-box armadillo repeat (PUB-ARM) proteins represent a type of E3 ubiquitin ligase. PUB-ARM proteins have various functions in plant development, plant defence and plant stress responses. The so far uncharacterised PUB-ARM proteins AtPUB18 and AtPUB19 are highly homologous to each other and regulated on the transcript level by ABA and NaCl. To investigate their physiological function, we isolated and characterised two Arabidopsis mutants and named them pub18-1 and pub19-1. However, these single mutants did not show any ABA- or salt-dependent phenotype. In contrast, pub18-1/pub19-1 double mutant seeds were less sensitive to ABA and NaCl inhibition of seed germination compared to wild-type seeds, indicating that both PUB-ARM proteins may coordinately function as regulatory components in ABA or salt inhibition of germination.

PMID: 21815976

2006

0.0

. 2006, 142(4):1664-1682

Heterologous expression and molecular and cellular characterization of CaPUB1 encoding a hot pepper U-Box E3 ubiquitin ligase homolog

Cho S , Chung H , Ryu M

Department of Biology, College of Science, Yonsei University, Seoul 120-749, Korea.

The U-box motif is a conserved domain found in the diverse isoforms of E3 ubiquitin ligase in eukaryotes. From water-stressed hot pepper (Capsicum annuum L. cv Pukang) plants, we isolated C. annuum putative U-box protein 1 (CaPUB1), which encodes a protein containing a single U-box motif in its N-terminal region. In vitro ubiquitination and site-directed mutagenesis assays revealed that CaPUB1 possessed E3 ubiquitin ligase activity and that the U-box motif was indeed essential for its enzyme activity. RNA gel-blot analysis showed that CaPUB1 mRNA was induced rapidly by a broad spectrum of abiotic stresses, including drought, high salinity, cold temperature, and mechanical wounding, but not in response to ethylene, abscisic acid, or a bacterial pathogen, suggesting its role in the early events in the abiotic-related defense response. Because transgenic work was extremely difficult in hot pepper, in this study we overexpressed CaPUB1 in Arabidopsis (Arabidopsis thaliana) to provide cellular information on the function of this gene in the development and plant responses to abiotic stresses. Transgenic Arabidopsis plants that constitutively expressed the CaPUB1 gene under the control of the cauliflower mosaic virus 35S promoter had markedly longer hypocotyls and roots and grew more rapidly than the wild type, leading to an early bolting phenotype. Microscopic analysis showed that 35S::CaPUB1 roots had increased numbers of small-sized cells, resulting in disordered, highly populated cell layers in the cortex, endodermis, and stele. In addition, CaPUB1-overexpressing plants displayed increased sensitivity to water stress and mild salinity. These results indicate that CaPUB1 is functional in Arabidopsis cells, thereby effectively altering cell and tissue growth and also the response to abiotic stresses. Comparative proteomic analysis showed that the level of RPN6 protein, a non-ATPase subunit of the 26S proteasome complex, was significantly reduced in 35SCaPUB1 seedlings as compared to the wild type. Pull-down and ubiquitination assays demonstrated that RPN6 interacted physically with CaPUB1 and was ubiquitinated in a CaPUB1-dependent manner in vitro. Although the physiological function of CaPUB1 is not yet clear, there are several possibilities for its involvement in a subset of physiological responses to counteract dehydration and high-salinity stresses in transgenic Arabidopsis seedlings.

PMID: 17041029

2008

0.0

... 蒺藜苜蓿是豆科苜蓿属草本^[15] ...

2014

0.0

. 2014, 23(1):239-247 DOI:10.11686/cyxb20140129

马铃薯和拟南芥GAPC酶基因的克隆及分

邹雪, 张烨, 吴明阳

College of Agronomy,Sichuan Agricultural University,Chengdu 611130,China

Cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPC) takes part in glycolysis and is a classical housekeeping enzyme in both prokaryotes and eukaryotes. Increasing evidence indicates that GAPC is involved in various plant abiotic and biotic stress responses such as salt,low phosphate,reactive oxygen species (ROS) and heat in phytophthora infestans. The AtGAPC 2 gene has been found in responses to low phosphate and osmotic stress in Arabidopsis . Analysis of sequences and protein structure of GAPC could help in studies of the function and mechanisms of GAPC acting in plant stress resistance. The fragments encoding GAPC were obtained from Solanum tuberosum (StGAPC) and Arabidopsis thialiana ( AtGAPC 2) seedlings by reverse transcription polymerase chain reaction (RT-PCR). The CDS lengths of StGAPC and AtGAPC 2 were 1017 bp,encoding a protein subunit of 338 amino acids. Bioinformatics analysis demonstrated that the StGAPC and AtGAPC 2 had 84% similarity in nucleotide sequence and 92% similarity in amino acid sequence. Molecular weights of the proteins were 36.65 and 36.91 kDa,the PI (theoretical isoelectric point) were 6.34 and 6.67,and both of them had stable protein structures. The 3D structures of StGAPC and AtGAPC 2 were predicted by homology comparative modeling in the Swiss-Model,and showed that the 3D structures were highly similar to Oryza sativa GAPDH C chain (2e5rC). Predicted proteins contained conserved GAPC domains which included an NAD binding domain constructed by βαβαβ Rossmann coil and a catalyzed domain in the saddle super secondary structure composed of seven β foldings. CDS sequences of GAPC showed that the same family of plants has a high homology attributed to the same branch in the evolutionary tree and this evolutionary relationship corresponded to the existing classification system. pBI121 was constructed as a base vector and the gene expression vector in the plant was driven by a CaMV35S promoter. The recombinant vector was introduced into Agrobacterium strain EHA105 and positive clones were determined by PCR. This research is a basis to obtain transgenic materials and for further study on the function of GAPC.

胞质三磷酸甘油醛脱氢酶(GAPC)是糖酵解中的关键酶,近年的研究表明其对盐、低温、高温、氧化、高渗、低磷等多种逆境胁迫均有响应。本实验采用RT-PCR,分别从马铃薯和拟南芥中克隆GAPC的编码区序列(CDS),并作生物信息学比较,同时构建2个基因的植物表达载体。结果表明, StGAPC 和 AtGAPC 2的CDS长度均为1017 bp,相似性84%,编码338个氨基酸,相似性为92%。蛋白分子量分别为36.65和36.91 kDa,理论等电点为6.34和6.67,均属稳定蛋白;三级结构预测表明两者和水稻(2e5rC)GAPC蛋白结构相似,具有GAPC保守功能域。多种植物 GAPC 基因的序列进化分析表明同科属植物的同源性较高可归于同一分支,与现有的分类系统相对应。以pBI121为基础载体,构建了由CaMV35S启动 GAPC 基因的植物表达载体,导入农杆菌EHA105,经PCR检测确定获得阳性克隆。本研究为获得转 GAPC 基因的材料并进一步研究GAPC在逆境中的功能奠定基础。

... 取蒺藜苜蓿叶片组织100 mg,按照北京全式金TransZol Plus RNA Kit(ER501)试剂盒说明步骤提取总RNA^[16] ...

... 0软件中选择邻近结合法(neighbor-joining)构建进化树,分析MtPUB4编码的氨基酸序列物种进化关系^[16] ...

... 通过构建系统进化树结果表明,它们与同科属植物同源性很高,和其他科属的植物同源性较低,这与其他学者在其他物种间同源性分析结论基本一致^[16,27],说明同源关系较近的植物中基因保守性更强 ...

2001

0.0

... 根据实验所得Ct值,利用Livak等^[17]报道的fold change=2^{-#cod#x00394 ...}

2006

0.0

. 2006, 7(8):509-522

The E3 ubiquitin ligase gene family in plants: regulation by degradation

Mazzucotelli , Belloni , Marone

C.R.A.-Experimental Institute for Cereal Research, Section of Foggia, S.S. 16 km 675, 71100 Foggia, Italy.

The regulation of protein expression and activity has been for long time considered only in terms of transcription/translation efficiency. In the last years, the discovery of post-transcriptional and post-translational regulation mechanisms pointed out that the key factor in determining transcript/protein amount is the synthesis/degradation ratio, together with post-translational modifications of proteins. Polyubiquitinaytion marks target proteins directed to degradation mediated by 26S-proteasome. Recent functional genomics studies pointed out that about 5% of Arabidopsis genome codes for proteins of ubiquitination pathway. The most of them (more than one thousand genes) correspond to E3 ubiquitin ligases that specifically recognise target proteins. The huge size of this gene family, whose members are involved in regulation of a number of biological processes including hormonal control of vegetative growth, plant reproduction, light response, biotic and abiotic stress tolerance and DNA repair, indicates a major role for protein degradation in control of plant life.

PMID: 18369404

... 有学者认为ARM结构域蛋白质在植物生长发育和激素应答等方面起重要作用^[9,18] ...

2014

0.0

... 在某些植物U-box蛋白中,如拟南芥中ARC1(Arm Repeat Containing 1),其C端含有一个6~7个ARM重复结构功能域,中间含有一个U-box功能域,是SRK激酶下游信号的传递因子,能在柱头乳突细胞中特异性表达,能促进花粉水合、萌发和花粉管生长的雌性亲和因子^[19] ...

2015

0.0

. 2015, 11(2):e1004629- DOI:10.1371/journal.ppat.1004629

The RhoGAP SPIN6 associates with SPL11 and OsRac1 and negatively regulates programmed cell death and innate immunity in rice

Liu , Park C , He

State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China; Hunan Provincial Key Laboratory of Crop Germplasm Innovation and Utilization and College of Agronomy, Hunan Agricultural University, Changsha, Hunan, China.

The ubiquitin proteasome system in plants plays important roles in plant-microbe interactions and in immune responses to pathogens. We previously demonstrated that the rice U-box E3 ligase SPL11 and its Arabidopsis ortholog PUB13 negatively regulate programmed cell death (PCD) and defense response. However, the components involved in the SPL11/PUB13-mediated PCD and immune signaling pathway remain unknown. In this study, we report that SPL11-interacting Protein 6 (SPIN6) is a Rho GTPase-activating protein (RhoGAP) that interacts with SPL11 in vitro and in vivo. SPL11 ubiquitinates SPIN6 in vitro and degrades SPIN6 in vivo via the 26S proteasome-dependent pathway. Both RNAi silencing in transgenic rice and knockout of Spin6 in a T-DNA insertion mutant lead to PCD and increased resistance to the rice blast pathogen Magnaporthe oryzae and the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. The levels of reactive oxygen species and defense-related gene expression are significantly elevated in both the Spin6 RNAi and mutant plants. Strikingly, SPIN6 interacts with the small GTPase OsRac1, catalyze the GTP-bound OsRac1 into the GDP-bound state in vitro and has GAP activity towards OsRac1 in rice cells. Together, our results demonstrate that the RhoGAP SPIN6 acts as a linkage between a U-box E3 ligase-mediated ubiquitination pathway and a small GTPase-associated defensome system for plant immunity.

PMID: 25658451

... 在水稻中发现的SPL11^[20,21,22]、烟草中的ACER276^[23]、拟南芥中的AtPUB17等U-box基因都与植物抗病性有关^[24] ...

2008

0.0

. 2008, 20(6):1456-1469 DOI:10.1105/tpc.108.058610

SPIN1, a K homology domain protein negatively regulated and ubiquitinated by the E3 ubiquitin ligase SPL11, is involved in flowering time control in rice

Vega-Sanchez M , Zeng , Chen

Department of Plant Pathology, Plant Molecular Biology and Biotechnology Program, Ohio State University, Columbus, Ohio 43210, USA.

The rice (Oryza sativa) E3 ligase SPOTTED LEAF11 (SPL11) negatively regulates programmed cell death and disease resistance. We demonstrate here that SPL11 also regulates flowering via interaction with SPIN1 (for SPL11-interacting protein1), a Signal Transduction and Activation of RNA family member. SPIN1 binds RNA and DNA in vitro and interacts with SPL11 in the nucleus. Spl11 mutants have delayed flowering under long-day conditions. Spin1 overexpression causes late flowering independently of daylength; expression analyses of flowering marker genes in these lines suggested that SPIN1 represses flowering by downregulating the flowering promoter gene Heading date3a (Hd3a) via Hd1-dependent mechanisms in short days and by targeting Hd1-independent factors in long days. Both Spin1 and Spl11 are regulated diurnally in opposing phases. SPL11 negatively regulates Spin1 transcript levels, while SPIN1 also affects Spl11 expression. Moreover, we show that coincidence of high accumulation of Spin1 mRNA with the light in the morning and early evening is needed to repress flowering. SPIN1 is monoubiquitinated by SPL11, suggesting that it is not targeted for degradation. Our data are consistent with a model in which SPIN1 acts as a negative regulator of flowering that itself is negatively regulated by SPL11, possibly via ubiquitination.

PMID: 18586868

... 在水稻中发现的SPL11^[20,21,22]、烟草中的ACER276^[23]、拟南芥中的AtPUB17等U-box基因都与植物抗病性有关^[24] ...

2012

0.0

. 2012, 160(1):28-37 DOI:10.1104/pp.112.199430

The U-box E3 ligase SPL11/PUB13 is a convergence point of defense and flowering signaling in plants

Liu , Li , Ning

State Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.

PMID: 22659522

... 在水稻中发现的SPL11^[20,21,22]、烟草中的ACER276^[23]、拟南芥中的AtPUB17等U-box基因都与植物抗病性有关^[24] ...

2006

0.0

... 在水稻中发现的SPL11^[20,21,22]、烟草中的ACER276^[23]、拟南芥中的AtPUB17等U-box基因都与植物抗病性有关^[24] ...

2015

0.0

. 2015, 66(11):3189-3199 DOI:10.1093/jxb/erv128

McLellan H, Boevink P C, et al. U-box E3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans

Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan, Hubei, 430070, China and the National Centre for Vegetable Improvement (Central China), Huazhong Agricultural University, Wuhan, Hubei, 430070, China; Division of Plant Sciences, University of Dundee, James Hutton Institute (JHI), Errol Road, Invergowrie, Dundee DD2 5DA, UK Dundee Effector Consortium, James Hutton Institute, Errol Road, Invergowrie, Dundee DD2 5DA, UK.

Ubiquitination regulates many processes in plants, including immunity. The E3 ubiquitin ligase PUB17 is a positive regulator of programmed cell death (PCD) triggered by resistance proteins CF4/9 in tomato. Its role in immunity to the potato late blight pathogen, Phytophthora infestans, was investigated here. Silencing StPUB17 in potato by RNAi and NbPUB17 in Nicotiana benthamiana by virus-induced gene silencing (VIGS) each enhanced P. infestans leaf colonization. PAMP-triggered immunity (PTI) transcriptional responses activated by flg22, and CF4/Avr4-mediated PCD were attenuated by silencing PUB17. However, silencing PUB17 did not compromise PCD triggered by P. infestans PAMP INF1, or co-expression of R3a/AVR3a, demonstrating that not all PTI- and PCD-associated responses require PUB17. PUB17 localizes to the plant nucleus and especially in the nucleolus. Transient over-expression of a dominant-negative StPUB17(V314I,V316I) mutant, which retained nucleolar localization, suppressed CF4-mediated cell death and enhanced P. infestans colonization. Exclusion of the StPUB17(V314I,V316I) mutant from the nucleus abolished its dominant-negative activity, demonstrating that StPUB17 functions in the nucleus. PUB17 is a positive regulator of immunity to late blight that acts in the nucleus to promote specific PTI and PCD pathways.

PMID: 25873665

... 在水稻中发现的SPL11^[20,21,22]、烟草中的ACER276^[23]、拟南芥中的AtPUB17等U-box基因都与植物抗病性有关^[24] ...

2001

0.0

. 2001, 106(3):343-354

Garcia-Martinez J L, et al. Gibberellins signal nuclear import of PHOR1, a photoperiod-responsive protein with homology to Drosophila armadillo

Amador , Monte

Departament de Genètica Molecular, Institut de Biologia Molecular de Barcelona-CSIC, Jordi Girona, 18-26, 08034 Barcelona, Spain.

S. tuberosum ssp. andigena potato plants require short days (SD) for tuberization. We have isolated PHOR1 (photoperiod-responsive 1), which shows upregulated expression in induced leaves (SD). PHOR1 encodes an arm repeat protein with homology to the Drosophila segment polarity protein armadillo. Antisense inhibition of PHOR1 produces a semidwarf phenotype similar to that of GA-deficient plants, and the antisense lines show reduced GA responsiveness combined with a higher endogenous GA content than wild-type plants. Feedback regulation of GA biosynthetic genes is also altered in these lines. Conversely, transgenic lines overexpressing PHOR1 show an enhanced response to GA. GA application induces rapid migration of PHOR1-GFP protein to the nucleus. Thus, PHOR1 appears to be a general component of GA signaling pathways that relocalizes to the nucleus in the presence of GA.

PMID: 11509183

... 马铃薯(Solanum tuberosum)中发现的U-box基因PHOR1(photoperiod-responsive1)参与GA信号转导,是GA信号转导途径中的正调控因子^[25] ...

2003

0.0

... 烟草中的NtPUB4参与了植物发育和细胞分裂素信号转导^[26] ...

... Kim等^[26]研究认为,烟草的NtPUB4参与CHRK1介导的植物发育及细胞分裂素信号转导过程 ...

... 而CHRK1与SRK类受体激酶聚类在一起,推测NtPUB4与ARC1功能相似^[26] ...

2012

0.0

. 2012, 21(3):106-116 DOI:10.11686/cyxb20120314

马铃薯SGAs合成代谢途径末端SGT酶基因克隆及序列分

牛继平, 张金文, 王旺田

糖苷生物碱（ＳＧＡｓ）是一类存在于茄科植物和百合科植物的重要次生代谢物，与植物抗逆性和产品品质有密切关系，同时在医药上具有广泛的药理学活性。茄啶：糖基转移酶（ＳＧＴ）为ＳＧＡｓ合成代谢途径末端关键酶，研究其基因结构及其编码的酶蛋白特性，对于调控植物体内ＳＧＡｓ合成及其通过微生物发酵生产ＳＧＴｓ有重要的作用和意义。通过生物信息学分析方法预测３个糖基转移酶ｃＤＮＡ编码的酶蛋白结构和特性。以马铃薯块茎表皮总ＲＮＡ为模板，采用ＲＴ－ＰＣＲ的方法扩增出３个糖基转移酶（ＳＧＴ１、ＳＧＴ２和ＳＧＴ３）基因片段并克隆到ｐＭＤＲ１９Ｔ载体。阳性克隆经ＰＣＲ鉴定后进行测序，序列分析结果表明，克隆到的 sgt １、 sgt ２和 sgt ３三个同源的序列片段（１４６７～１５１８ｂｐ），编码４８８～５０５个氨基酸；所得序列与ＧｅｎＢａｎｋ中注册的高等植物ＳＧＴ酶基因核苷酸序列（Ｕ８２３６７．２、ＤＱ２１８２７６．１和ＤＱ２６６４３７）的同源性均在９９．１２％以上，氨基酸同源性达到９９％以上，３个基因都具有ＵＤＰＧ糖基转移酶保守结构域及许多重要功能位点；ＰＩ为５．５２～５．６２。三级结构预测表明，该氨基酸同糖基转移酶单体结构模型相似，都为糖基转移酶家族成员，具有合成类固醇糖苷生物碱的功能，基因序列已注册到Ｇｅｎ Ｂａｎｋ，序列注册号分别为： sgt １，ＪＮ６９５００５； sgt ２，ＪＮ６９５００６； sgt ３，ＨＭ１８８４４７。

2008

0.0

. 2008, 147(4):2084-2095 DOI:10.1104/pp.108.123380

Interactions between the S-domain receptor kinases and AtPUB-ARM E3 ubiquitin ligases suggest a conserved signaling pathway in Arabidopsis

Samuel M , Mudgil , Salt J

Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, Canada M5S 3B2.

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses.

PMID: 18552232

... 前人研究表明U-box E3连接酶可调控植物多个生理代谢途径,Samuel等^[28]研究发现,拟南芥中的U-box/ARM蛋白家族与拟南芥SD1受体蛋白激酶亚家族相互结合,通过酵母双杂交实验表明,两个受体结合可使U-box/ARM蛋白的ARM功能域磷酸化,从而参与调控植物生理过程 ...

... Samuel等^[28]在拟南芥中研究发现,一些U-box/ARM基因受到NaCl胁迫诱导表达,基因表达量会上调 ...

2012

0.0

. 2012, 22(4):649-660 DOI:10.1038/cr.2012.28

Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA

Zhou , Liu , Li

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, China.

Photoperiod- and thermo-sensitive genic male sterility (PGMS and TGMS) are the core components for hybrid breeding in crops. Hybrid rice based on the two-line system using PGMS and TGMS lines has been successfully developed and applied widely in agriculture. However, the molecular mechanism underlying the control of PGMS and TGMS remains obscure. In this study, we mapped and cloned a major locus, p/tms12-1 (photo- or thermo-sensitive genic male sterility locus on chromosome 12), which confers PGMS in the japonica rice line Nongken 58S (NK58S) and TGMS in the indica rice line Peiai 64S (PA64S, derived from NK58S). A 2.4-kb DNA fragment containing the wild-type allele P/TMS12-1 was able to restore the pollen fertility of NK58S and PA64S plants in genetic complementation. P/TMS12-1 encodes a unique noncoding RNA, which produces a 21-nucleotide small RNA that we named osa-smR5864w. A substitution of C-to-G in p/tms12-1, the only polymorphism relative to P/TMS12-1, is present in the mutant small RNA, namely osa-smR5864m. Furthermore, overexpression of a 375-bp sequence of P/TMS12-1 in transgenic NK58S and PA64S plants also produced osa-smR5864w and restored pollen fertility. The small RNA was expressed preferentially in young panicles, but its expression was not markedly affected by different day lengths or temperatures. Our results reveal that the point mutation in p/tms12-1, which probably leads to a loss-of-function for osa-smR5864m, constitutes a common cause for PGMS and TGMS in the japonica and indica lines, respectively. Our findings thus suggest that this noncoding small RNA gene is an important regulator of male development controlled by cross-talk between the genetic networks and environmental conditions.

PMID: 22349461

... 目前,利用这种现象人工筛选突变体,通过两系配套法培育不育系已在杂交作物的生产上成功应用^[29] ...

2008

0.0

2012

0.0

... 该过程首先是靶蛋白被泛素分子标记,然后被蛋白酶体识别后降解^[2] ...

2015

0.0

. 2015, 4:1-8, 28

E3泛素连接酶调控植物抗病分子机理研究进

杨玖霞, 张浩, 王志龙

2008

0.0

2015

0.0

. 2015, 24(8):130-141 DOI:10.11686/cyxb2015102

蒺藜苜蓿全基因组中U-box基因家族的筛选与特征分

郑兴伟, 邵麟惠, 李聪

Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

... 目前,有关蒺藜苜蓿U-box基因家族的研究尚未见报道^[12] ...

... 0软件邻近法程序构建系统进化树^[12] ...

2008

0.0

... 蒺藜苜蓿是豆科苜蓿属草本^[15] ...

2014

0.0

. 2014, 23(1):239-247 DOI:10.11686/cyxb20140129

马铃薯和拟南芥GAPC酶基因的克隆及分

邹雪, 张烨, 吴明阳

College of Agronomy,Sichuan Agricultural University,Chengdu 611130,China

... 取蒺藜苜蓿叶片组织100 mg,按照北京全式金TransZol Plus RNA Kit(ER501)试剂盒说明步骤提取总RNA^[16] ...

... 0软件中选择邻近结合法(neighbor-joining)构建进化树,分析MtPUB4编码的氨基酸序列物种进化关系^[16] ...

2012

0.0

. 2012, 21(3):106-116 DOI:10.11686/cyxb20120314

马铃薯SGAs合成代谢途径末端SGT酶基因克隆及序列分

牛继平, 张金文, 王旺田